1. Field of the Invention
This invention is related to the field of probe-based detection, analysis, identification and/or quantitation of organisms and/or cells.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, quantitation and/or analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, nucleic acid probe-based assays have been slow to achieve commercial success. This lack of commercial success is, at least partially, the result of difficulties associated with specificity, sensitivity and/or reliability.
Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide that can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. No. 5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). Being a non-naturally occurring molecule, unmodified PNA is not known to be a substrate for the enzymes that are known to degrade peptides or nucleic acids. Therefore, PNA should be stable in biological samples, as well as have a long shelf-life. Unlike nucleic acid hybridization, which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions that strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). Because of their unique properties, it is clear that PNA is not the equivalent of a nucleic acid in either structure or function. Consequently, PNA probes need to be evaluated for performance and optimization to thereby confirm whether they can be used to specifically and reliably detect a particular nucleic acid target sequence, particularly when the target sequence exists in a complex sample such as a cell, tissue or organism.
A probe-based assay, whether performed using a nucleic acid or non-nucleic acid probe (e.g. a PNA), typically involves a multistep procedure wherein reagents are sequentially added to the sample containing the cells or organism and then removed by centrifugation (i.e. pelleting of the cells followed by a decanting of the reagents) and/or associated washing steps. In many cases, the organisms or cells of the sample are first grown in a medium to increase their total number and are then isolated from the growth medium before being treated with the assay reagents. At a minimum, the assay typically involves a step for the fixation of the cells or organisms and a step for the treatment of the cells or organisms with a molecular probe to thereby render the organisms or cells detectable. Applicants however, are not aware of any existing assay for the determination of cells or organisms that operates without separating the fixing reagent prior to the step of determination. Moreover, when cells are grown in a medium, Applicants are not aware of any assay for the determination of cells or organisms that operates without separating growth medium, fixing reagent and/or excess molecular probe prior to the step of determination.